mmu-miR-1963 negatively regulates the ameloblast differentiation of LS8 cell line by directly targeting Smoc2 3'UTR.

RUNX2 is a key regulator of osteogenic differentiation and odontoblastic differentiation. RUNX2 mutations could cause Cleidocranial dysplasia (CCD; OMIM119600), which is featured by abnormal development of bone and teeth. By using microRNA array, we identified a large number of microRNAs that showed different expression between wild-type Runx2 group and mutant groups. The aim of this study is to find out the effect of mmu-miR-1963, which was downregulated in all mutant Runx2 groups, on the ameloblast differentiation of LS8 cells. qPCR and Western Blot results showed the suppressive effect of mmu-miR-1963 on ameloblast differentiation of LS8 cell line. We further confirmed Smoc2 as one direct target of mmu-miR-1963. For the first time, we showed that mmu-miR-1963 could regulate the ameloblast differentiation of LS8 by targeting Smoc2. This study suggests the suppressive role of mmu-miR-1963 on ameloblast differentiation of LS8 via directly targeting the 3'UTR of Smoc2. We also demonstrated that Smoc2 itself could promote the ameloblast differentiation of LS8 for the first time. Our results indicate a novel explanation to the enamel hypoplasia phenotype in part of CCD patients.

Mutant Runx2 regulates amelogenesis and osteogenesis through a miR-185-5p-Dlx2 axis.

Regulation of microRNAs (miRNA) has been extensively investigated in diseases; however, little is known about the roles of miRNAs in cleidocranial dysplasia (CCD). The aim of the present study was to investigate the potential involvement of miRNAs in CCD. In vitro site-directed mutagenesis was performed to construct three mutant Runx2 expression vectors, which were then transfected into LS8 cells and MC3T3-E1 cells, to determine the impact on amelogenesis and osteogenesis, respectively. miRCURY LNA miRNA microarray identify miR-185-5p as a miRNA target commonly induced by all three Runx2 mutants. Real-time quantitative PCR was applied to determine the expression of miR-185-5p and Dlx2 in samples. Dual-luciferase reporter assays were conducted to confirm Dlx2 as a legitimate target of miR-185-5p. The suppressive effect of miR-185-5p on amelogenesis and osteogenesis of miR-185-5p was evaluated by RT-PCR and western blot examination of Amelx, Enam, Klk4, and Mmp20 gene and protein expression, and by Alizarin Red stain. We found that mutant Runx2 suppressed amelogenesis and osteogenesis. miR-185-5p, induced by Runx2, suppressed amelogenesis and osteogenesis. Furthermore, we identified Dlx2 as direct target of miR-185-5p. Consistently, Dlx2 expression was inversely correlated with miR-185-5p levels. This study highlights the molecular etiology and significance of miR-185-5p in CCD, and suggests that targeting miR-185-5p may represent a new therapeutic strategy in prevention or intervention of CCD.

Restorative treatment strategies for patients with cleidocranial dysplasia.

OBJECTIVE: To develop a suitable treatment strategy for patients with cleidocranial dysplasia (CCD) who miss the optimal early treatment stage. MATERIALS AND METHODS: This study enrolled 15 patients with CCD who had all missed the optimal treatment stage and were diagnosed with CCD through clinical examinations and genetic tests. Based on the chief complaints and requirements of the patients, three different therapeutic schedules were devised for these patients. Schedules I (periodontal and endodontic treatments) and II (periodontal, endodontic and prosthodontic treatments) were used for patients with low requirements, whereas Schedule III (multidisciplinary strategy, including periodontal, endodontic, surgical, orthodontic and prosthodontic treatments) was used for patients with high requirements. RESULTS: Schedules I, II and III were used in five, seven and three patients, respectively. Schedule III treatments produced the best outcomes in terms of occlusion and esthetics. CONCLUSIONS: Schedule III based on a comprehensive multidisciplinary therapy is an ideal restorative therapeutic strategy and can achieve good outcomes for patients with CCD who missed the optimal treatment stage.

A novel non-stop mutation in MSX1 causing autosomal dominant non-syndromic oligodontia.

Oligodontia, which is the congenital absence of six or more permanent teeth, excluding the third molars, may contribute to masticatory dysfunction, speech alteration, aesthetic problems and malocclusion. Msh homeobox 1 (MSX1) was the first gene identified as causing non-syndromic oligodontia. In this study, we identified a novel heterozygous non-stop mutation (c.910_911dupTA, p.*304Tyrext*48) in MSX1 in a Chinese family with autosomal dominant non-syndromic oligodontia. This novel mutation substitutes the stop codon with a tyrosine residue, potentially adding 48 amino acids to the C-terminus of MSX1. Further in vitro study found that mutant MSX1 could be expressed but had lost its ability to enter the nucleus. This is the first report indicating that a non-stop mutation in MSX1 is responsible for oligodontia. This study broadens the mutation spectrum for MSX1 and provides a new way to clarify the mechanism of MSX1 in tooth agenesis.

Novel missense mutations in the AXIN2 gene associated with non-syndromic oligodontia.

OBJECTIVE: Oligodontia, which is the congenital absence of six or more permanent teeth excluding third molars, may contribute to masticatory dysfunction, speech alteration, aesthetic problems and malocclusion. To date, mutations in EDA, AXIN2, MSX1, PAX9, WNT10A, EDAR, EDARADD, NEMO and KRT 17 are known to associate with non-syndromic oligodontia. The aim of the study was to search for AXIN2 mutations in 96 patients with non-syndromic oligodontia. DESIGN: We performed mutation analysis of 10 exons of the AXIN2 gene in 96 patients with isolated non-syndromic oligodontia. RESULTS: We identified two novel missense mutations (Exon 3 c.923C>T and Exon 11 c.2490G>C) in two patients. One mutation (c.923C>T) results in a Thr308Met substitution and the other mutation (c.2490G>C) results in a Met830Ile substitution. CONCLUSIONS: This is the first report indicating that mutations in AXIN2 are responsible for oligodontia in the Chinese population. Our findings indicate that AXIN2 can be regarded as a candidate gene for mutation detection in individuals with non-syndromic oligodontia in the Chinese population.